Anti phospho histone h2ax ser139

Western blotting was carried out by standard protocols. Anti-Brca1 (sc-28234, Santa Cruz, CA, USA) and anti-Phospho-Histone H2A.X (Ser139) (2577S, Cell Signaling Technology, Beverly, MA, USA) were used as primary antibodies. GAPDH (10494-1-AP, Proteintech, Chicago, IL, USA) and ß-Tublin (sc-9104, Santa Cruz, CA, USA) served as loading controls. The protein bands were detected using an Enhanced Chemiluminescence Detection System (Millipore, Billerica, MA, USA). Each experiment was repeated at least three times.

The 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), crystal violet, propidium iodide (PI), RNAse, cell lysis buffer, dimethyl sulfoxide (DMSO), phenylmethylsulfonyl fluoride (PMSF), protease and phosphatase inhibitor cocktails, and trypan blue were purchased from Sigma-Aldrich (St. Louis, MO, USA). The mouse monoclonal anti-PARP antibody, anti-phospho-histone H2A.X (Ser139), mouse monoclonal anti-cdc2 (CDK1) antibody, anti-phospho-cdc2 (Thr161) antibody, and mouse monoclonal anti-cyclin D3 were acquired from Cell Signaling Technology, Inc. (Beverly, MA, USA). The monoclonal anti-β-actin antibody was acquired from Sigma-Aldrich (St. Louis, MO, USA). The monoclonal anti-cyclin B1, goat anti-mouse IgG-horseradish peroxidase (HRP), and goat anti-rabbit IgG-HRP antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).
The phycoerythrin (PE)-conjugated anti-CD133/2 (293C3) antibody was purchased from Miltenyi Biotec (Bologna, Italy). The fluorescein isothiocyanate (FITC)-conjugated anti-CD326 (EpCAM) antibody was purchased from BD (BD, Becton-Dickinson Biosciences, San Jose, CA, USA). The Live/Dead Fixable Far Red Dead cell stain kit (Reactive Dye) was acquired from Thermo Fisher Scientific (Waltham, MA, USA).

Doxorubicin (#5927, cell signaling), hydroxytyrosol (H4291 Sigma-Aldrich, Missouri, MO, USA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) (Sigma-Aldrich). Antibodies: anti-cleaved caspase-3 (#9662), anti-SOD2 (#13194), anti-Bax (#2774), anti-Bcl-2 (#15071), anti-phospho-p38 MAPK (#9211), anti-p-38 (#9212), anti-phospho-histone H2AX (Ser139) (#80312), Anti-GAPDH (#97166), anti-tubulin (#2146) (Cell Signalling Technology, Danvers, MA, USA). Secondary antibodies: anti-mouse IgG (#7076), anti-rabbit IgG (#7074) (Cell Signalling Technology).

Cells for each condition were treated in vitro with 1 μmol/L cisplatin for 72 h to assess DNA damage. Subsequently, treated cells and negative controls were fixed in PFA 4% for 15 min at RT and then permeabilized in ice-cold 90% methanol for 10 min in ice. Cells were washed once in BD PERM/WASH buffer 1× (Becton Dickinson, BD, Franklin Lakes, NJ, USA) to remove methanol and then resuspended in 100 µL of 1:100 diluted Anti-Phospho-Histone H2AX (Ser139) (Cell Signaling, Beverly, MA, USA). After 1 h of primary antibody incubation, cells were stained for 30 min with AlexaFluor 488 conjugated secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA). Cells were then washed once in BD PERM/WASH buffer 1X and resuspended in 400 µL of BD PERM/WASH buffer 1X for flow cytometric analysis. To assess apoptosis, cisplatin-treated cells as reported above and controls were stained following the kit protocol PE Annexin V Apoptosis Detection Kit I (RUO) (Becton Dickinson, BD, Franklin Lakes, NJ, USA).

HCT116 and SF-TY cells were seeded into CellCarrier-96 plates (PerkinElmer) at 5 × 10³ cells/well, and after 24 h of incubation, the cells were divided into four groups (control, ATMi, Chk1i, and combined treatment) and cultured for 48 h. Cells were fixed for 30 min at room temperature in 4% paraformaldehyde, washed with PBS, and then permeabilized for 5 min in PBS containing 0.2% Triton X-100. After washing with PBS, cells were blocked with PBS containing 3% BSA, 0.1% glycine, and 0.1% NaN₃ for 1 h and then incubated overnight with the primary antibodies, anti-phospho-histone H2A.X (Ser139) (catalog number 9718; Cell Signaling Technology) and anti-53BP1 (catalog number 4937; Cell Signaling Technology). After washing with PBS, the cells were incubated with secondary antibody (catalog number A11070; Invitrogen) and 1 μg/mL of 4,6-diamidino-2-phenylindole (DAPI; DOJINDO) for 1 h at room temperature. An Operetta high-content imaging system (PerkinElmer, Waltham, MA, USA) for phospho-histone H2A.X and an LSM800 confocal microscope (Carl Zeiss, Oberkochen, Germany) for 53BP1 foci were used to observe the stained samples. Harmony^® high-content analysis software (Harmony software) (PerkinElmer) was used to determine the number of positive cells. The antibodies used in immunofluorescence staining were diluted in PBS supplemented with 0.2% Triton X-100.